8. Bacterial Genetics And Recombination

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mutation

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change in the nucleotide in the genome

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Genetic recombination

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genetic rearrangements in the genome

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Point Mutation

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involves a single base mutation

1. missense

2. nonsense

3. silent

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Missense

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results in a faulty protein

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nonsense

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results in an incomplete protein

( "non" -> not)

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silent

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point mutation that has no effect as the protein is the same because of the wobble

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agents that induce mutations in DNA

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1. chemical mutagen

2. radiation

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Chemical mutagen

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1. base analog

2. intercalative dyes

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base analog

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susbtitute for bases in DNA. pair with the wrong base induce a mutation

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intercalative

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ethidium bromide

causes abnormal DNA confirmation and results in the insertions/deletions (used to stain DNA)

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Ionizing radiation

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X-rays, cosmic rays, gamma rays. generate hydroxyl radicals that can mutate the DNA

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nonionizing radiation

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UV light. bases of DNA absorb UV light and generate altered DNA. some of the base pairs can absorb the UV light and cause the bases to mutate and get addition

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DNA Repair

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DNA repair systems act to repair damaged DNA prior

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SOS repair system

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activated by DNA damage

usually repressed by LeXA--> LexA is inactivated by RecA when DNA damage occurs

- induced error-prone DNA, called MUTASES

* E.coli has the SOS repair system

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Mechanism of SOS Repair System

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Lex A represses RecA in a normal cell with ni DNA damage

2. when DNA damage occurs, RecA binds to single-stranded DNA and s DNA damage accumulates, more RecCA will be boound to the DNA to repair damage

3. RecA then releases DNA in unbound form and no longer causes autoprotelysis of cellular levels of LexA rise to normal again, which shot down the expression of the SOS regulation genes

4. error-prone system-cause mutases

5. RuvABC- mediates branch migration and resolve Holliday junction created during homologous recombination in bacteria. so it s critical to bacteria DNA repair

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Ames Test of Mutagency

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it is used to determine the mutagenicity of a compound related to the carcinogenicity (high correlation)

- use a mutant strain and screen for mutations that repair the defect (revertants)

-IMPORTANT- THAT THE MUTATION IS POINT MUTATION

- ALSO HAS A MUTATION IN THE ERROR-PROOF DNA REPAIR MECHANISM

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mechanism of Ames Test`

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1. a histidine mutant of salmonella grown on medium without histodine, cells do not grow

2. a filter containing water or a test compound, revealing it to be a mutagen

3. 3/4 of chemicals that are positive in the Ames test are found to be rodent carcinogens

4. *easy and rapid to do this test

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genetic recombination

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a physical exchange of genetic material between genetic elements

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Homologous recombination

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exchange between homologous DNA sequences from different sources

requires RecA protein. homologs of RecA have been found in all species

*rad 51 is very similar

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The Holliday junction

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accounts for DNA exchange in YEAST

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4 ways to exchange genetic Material

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1. transformation

2. transduction

3. conjugation

4. nanotubes

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Transformation

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Definition: cell uptake DNA from environment

Competence: ability of cell to uptake DNA from environment

Mechanism:

1. DNA from environment binds to DNa binding protein on cell membrane

2. DNA taken up and can be degraded by nuclease as it enters cell

3. bound by comptence specific ssDNA binding proteins in cytoplasm

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transduction (think of transducing particle viruses)

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Definition: transfers DNA from one cell to another cell by virus

generalized- transfers DNA from any region

Specialized - transfers DNA from specific region

transducing particle- phage containing bacterial DNA

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Generalized transduction Mxm

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1. virus infects cell

2. transducing particle formed

3. transducing particle infects new cell

4. Donor's DNA recombines with recipients genome

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Specialized transduction

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phage DNA inserts into host DNA (always in same location- why it is specialized)

2. becomes induced and excises out of chromosome

3. makes more phage particles

sometimes

1. excise a piece of host DNA

2. packages host DNA into phage particle

3. transducing particle transfer the genes to new cell

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Conjugation

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DNA transfered by cell to cell contact

*bacterial sex

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conjugation mechanism

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1. donor cell contains conjugative plasmid which codes for sex pilus

2. pilus binds to receptor on recipient cell - pulls it closer

3. plasmid is copied by rolling circle replication and displaced strand is transferred (plasmid contains operon that codes for proteins involved in DNA transfer/ replication/ mating pair formation)

4. second strand synthesis occurs in recipient

Episomes- transfer of chromosomal genes

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rolling circle replication

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process of unidirectional replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA

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Episomes

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genetic element inside bacterial cells that can replicate independently of the host and also in association with a chromosome with which it becomes integrated

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Hfr ( high frequency of recombination)

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transfer entire DNA b/c plasmid is integrated into the chromosomes --> transfer of chromosomal genes

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F+ & F-

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strain has the F plasmid

- one strand is passed to daughter cell, copied, and then overall result is two F+ cells so it is important for drug resistance

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Donor cell (during conjugation)

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contains a conjugative plasmid

codes for a sex pilus

-pilus binds to receptor on recipient cell- pulls it closer

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Nanotubes

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small tubes linking different species

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Gene transfer in archaea

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archaea contain circular chromosome

-genetic manipulation of archaea lags behind bacteria

- most antibiotics do not affect archaea

no single species is a model organism for archaea

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why should we care about genetic exchange in bacteria ?

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plasmids often confer resistance to antibiotics

- virulence plasmids- can encode for toxins that promote disease

- horizontal gene transfer